Methylation of DNA is involved in both normal and abnormal cellular processes. For example, DNA methylation has been implicated in X-inactivation, genomic imprinting, and differential gene expression (such as by upregulation or silencing of genetic loci). DNA methylation plays a role in gene inactivation, cell differentiation, tumorigenesis, X-chromosome inactivation, and is required for mammalian development (Li, et al., Cell 69:915-926, 1992; Okano et al., Cell 99:247-57, 1999). In bacteria, methylation of cytosine and adenine residues plays a role in the regulation of DNA replication and DNA repair. DNA methylation has also been associated with increased risk of cancer, as well as cancer development itself.
Methylation of DNA is carried out by methylases (also known as methyltransferases). These enzymes are generally sequence-specific, and they can methylate both nucleic acid strands (in the case of DNA). Replication of these strands yields a hemi-methylated state which is recognized by a class of maintenance methylases capable of restoring full methylation to both strands.
Methylation can occur at all nucleotide residues, although in mammalian species, DNA methylation commonly occurs at cytosine residues, and more commonly at cytosine residues that lie next to a guanosine residue, i.e., at cytosine residues of a CG dinucleotide. CG dinucleotides in “CpG islands” remain methylation-free. CpG islands are rich in CG sites and are often found near coding regions within the genome (i.e., genes). About half of the genes in the human genome are associated with CpG islands. Importantly, the vast majority of CpG islands in the genome remain unmethylated in normal adult cells and tissues. Methylation of CpG islands is normally seen only on the inactive X-chromosome in females and at imprinted genes where it functions in the stable silencing of such genes. Strict control over the levels and distribution of DNA methylation are essential to normal animal development.
Alteration in DNA methylation is one manifestation of the genome instability characteristic of human tumors. A hallmark of human carcinogenesis is the loss of normal constraints on cell growth resulting from genetic alterations in the genes that control cell growth. The consequences of such mutations include the activation of positive growth signals and the inactivation of growth inhibitory signals. Identification of gene targets which when methylated lead to the loss of normal cell responses would be valuable. This would facilitate the diagnosis and treatment of disorders associated with abnormal methylation and any downstream events resulting therefrom.
The level of methylation of a nucleic acid can be determined using a number of techniques available in the art. Some methods of analysis involve the use of the chemical regent, bisulfite. Other methods for methylation analysis include methylation-sensitive restriction analysis, methylation-specific polymerase chain reaction (MSP), sequencing of bisulfite-modified DNA, Ms-SnuPE, and COBRA.
There is a serious need for improved methods for analyzing nucleic acid methylation status.
Fragmentation and labeling of nucleic acids are important for the analysis of nucleic acid sequences. For example, fragmentation and/or labeling are commonly required for detection of sequences by binding of a sample nucleic acid to complementary sequences immobilized on a surface, for example, on a microarray. Cleavage of sample nucleic acid into small fragments (e.g., 50-100 base pairs) facilitates diffusion of nucleic acid onto the surface, and may facilitate hybridization. It is known, for example, that steric and charge hindrance effects increase with the size of nucleic acids that are hybridized. Moreover, cleavage of sample nucleic acids into small fragments may ensure that two sequences of interest in the sample do not appear to bind to the same template nucleic acid simply by virtue of their proximity on the test nucleic acid. Cleavage of nucleic acids also facilitates detection of hybridized nucleic acid when, as in many detection methods, the size of the signal is proportional to the size of the bound fragment and thus, control of fragment size is desirable. Labeling of nucleic acids is necessary in many methods of nucleic acid analysis because there are presently few techniques for direct detection of unlabeled nucleic acid with the requisite sensitivity for analysis on chips. Methods for fragmenting and/or labeling nucleic acids are known in the art. See, e.g., U.S. Pat. Nos. 5,082,830; 4,996,143; 5,688,648; 6,326,142; and PCT Publication No. WO 02/090584, and references cited therein.
Immobilization of nucleic acids to create, for example, microarrays or tagged analytes, is useful for, e.g., detection and analysis of nucleic acids and tagged analytes. Methods for immobilizing nucleic acids are known in the art. See, e.g., U.S. Pat. Nos. 5,667,979; 6,077,674; 6,280,935; and references cited therein.
There is a serious need for improved methods for labeling and/or fragmenting and/or immobilizing nucleic acids to a surface (such as a microarray).
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.